The aim of this proposal is to study the biological mechanism of mother-to-child transmission (MTCT) of HIV using samples already collected from a well-characterized cohort of 860 HIV(+) pregnant Malawian women and their offspring. We hypothesize that: 1) the risk of MTCT is increased as a result of increased maternal-fetal microtransfusion, 2) maternal HIV diversity is a risk factor for MTCT, 3) heteroduplex tracking assays can be used to distinguish infants infected with HIV parenterally versus infants infected through mucosal membranes and 4) HIV is transmitted to infants through cell-associated virus. In a preliminary study, we measured placental alkaline phosphatase (PLAP), a placenta-impermeable high-molecular weight enzyme, in umbilical serum as a marker for maternal-fetal microtransfusion. In a nested case-cohort study of 204 mother-child pairs, elevated umbilical PLAP was found to be is a risk factor for intrapartum, but not intrauterine MTCT. Our first specific aim is to extend these observations by a) measuring PLAP in the umbilical cord sera from the full cohort, and b) developing and using a second method for maternal fetal microtransfusion based on genetic polymorphisms. In the second aim we will document maternal and infant viral diversity using heteroduplex tracking assays and assess the correlation between maternal diversity and MTCT; furthermore, we will use the infant HIV population diversity data, along with the data from Specific Aim 1, to determine if infant infections are a result of a mucosal exposure or direct inoculation via maternal contamination of fetal circulation. In the third Aim we will compare the profiles of maternal, umbilical and neonatal plasma^ and cell-associated virus to determine the relative importance of the two compartments to transmission. Thus, the results of this project will suggest a comprehensive model of the vector, the route, and the maternal factors associated with HIV vertical transmission.